Intervertebral disc (IVD) degeneration is a debilitating disorder implicated in the pathogenesis of low back pain with associated medical costs that can exceed $100 billion annually. The overall goal of the proposed research is to introduce novel therapeutic agents and strategies for use in a minimally invasive manner to limit degeneration, restore IVD structure, and reduce painful conditions of degenerative disc disease. Current therapies fail to integrate both structural repair and analgesia. Further, several analgesics are cytotoxic so that developing new therapeutic agents and strategies are a major research priority. The large vacuolated notochordal cells (NCs) in developing animals orchestrate patterning of the IVDs, vertebrae and surrounding spinal structures. Humans and other species that do not retain NCs into adulthood exhibit age related IVD degeneration and researchers have long sought to answer why NCs are lost in humans at young ages. The literature now indicates NCs are progenitor cells and suggests their early disappearance in humans is associated with their differentiation to small chondrocytic nucleus pulposus cells (SNPCs). For the first time, we have a bioreactor and culture methods capable of differentiating NCs into SNPCs so that we can derive therapies from NCs and explore mechanisms for their differentiation. The proposed studies provide a new paradigm for designing an integrated therapeutic intervention derived from trophic agents secreted by NCs to create structure and symptom modifying therapies capable of restoring IVD function and preventing discogenic pain by inhibition of neurovascular growth into the IVD. Aim 1 will determine microenvironment conditions capable of retaining NC phenotype, characterize the phenotypic stability of important genes and proteins of bioactive molecules produced by NCs, and isolate pathways involved in NC differentiation. Aim 2 is a series of descriptive and mechanistic studies that assess therapeutic potential of proteins secreted by NCs with dependent variables that focus on pain inhibition by limiting neurovascular invasion and promoting structural restoration. Aim 3 evaluates designed 'cocktail' treatments for their effects promoting anabolism and inhibiting discogenic pain or predictors of pain in human ex vivo organ culture models and rat in vivo discogenic pain models. This project is significant because of the translational potential to the highly clinically significant problem of discogenic back pain. The approach is innovative because it investigates factors important in developmental biology and introduces them for therapeutic effect using descriptive and mechanistic studies. We focus on therapeutic potential with mechanistic testing and screening studies using novel human organ culture and rat discogenic pain models. Innovation and significance are also high because determining optimal microenvironmental culturing conditions of NCs will help accelerate the growing body of research on these underexplored cells.